Vaccines for the prevention of congenital human cytomegalovirus (HCMV) infection continue to be a major public health priority. Vaccine studies in animal models of congenital infection provide insights into which types of vaccine strategies are likely to be useful in protecting the fetus. Among the cytomegaloviruses of small animals, the guinea pig cytomegalovirus (GPCMV) model is uniquely useful, because of the ability of this virus to cross the placenta and infect the pup, leading to infection and disease. In the initial grant period, we have demonstrated the usefulness of subunit vaccines in this model, using different expression strategies based on the GPCMV homologs of glycoprotein B (gB) and UL83 (GP83). In this competing continuation, we propose to test the hypotheses that vaccine-mediated protection can be augmented, using several expression strategies. First, we propose to examine strategies for improving the protective efficacy of DNA vaccination, compared to the gene gun approach, including cationic liposomes, and a "prime-boost" strategy using DNA vaccine and purified gB protein. Secondly, we will test the hypothesis that other viral glycoproteins, specifically the gM/gN complex, will be effective vaccines against congenital GPCMV infection, and that additional protection occurs when this vaccine is administered in combination with a gB vaccine. Finally, we will perform detailed assessments of the role of 2 cell-mediated immune targets, the UL83 (GP83) and the ie1 homologs, in the congenital infection model. In vivo assays will evaluate CD4+ and CD8+ cellular responses to these viral proteins, and CFSE studies will track T-cell trafficking in pregnant animals. Adoptive transfer studies will examine the role of immune T cells, in inbred animals, in protecting the maternal-placental-fetal unit from GPCMV infection. ELISPOT assays will be developed for the guinea pig cytokines, interferon gamma and IL-12. We anticipate that these subunit vaccine studies in this small animal model will clarify which strategies may be of value for vaccination against congenital HCMV infection.